HOT FIREPol® EvaGreen® HRM Mix (ROX)

A ready-to-use mix from Solis BioDyne formulated for High Resolution Melt (HRM) Analysis and qPCR using EvaGreen® dye. The 5x formulation contains HOT FIREPol® DNA Polymerase, ultrapure dNTPs, MgCl2, EvaGreen® dye and ROX dye, so the user only needs to add water, template and primers. HOT FIREPol® DNA Polymerase requires a 15 min hot-start activation at 95 °C, which prevents extension of non-specifically annealed primers and primer-dimers formed at low temperatures during reaction setup.

EvaGreen® is described as a superior DNA-binding dye for HRM, offering much less PCR inhibition, extreme stability and a non-mutagenic, non-cytotoxic profile. It is compatible with all common real-time PCR cyclers, where users can select the standard settings for SYBR® Green or FAM. The mix is supplied at 12.5 mM MgCl2, providing 2.5 mM MgCl2 at the 1x working concentration, and includes dNTPs (with dTTP, which improves reaction sensitivity and efficiency compared to dUTP), BSA and ROX.

Routine storage is at –20 °C; shipping and temporary storage at room temperature for up to 1 month have no detrimental effect on the quality of the mix. The recommended reaction setup uses 4 µl of 5x mix in a 20 µl total volume, with 80–250 nM of each primer and 1–50 ng/µl DNA template, made up to volume with PCR-grade water.